A fluorescence detection system utilizing fluorochrome as a labeling substance has hitherto been widely used in the fields of biochemistry and molecular biology. The use of this fluorescence detection system allows evaluations, for example, analysis of genetic sequence and genetic mutation and polymorphism, and separation and identification of proteins. Thus, the fluorescence detection system is utilized for development of medicines as an example.
As the above-described evaluation method utilizing fluorescent labeling, there is often used a method in which biological compounds, such as proteins, are distributed in a gel by electrophoresis and the distribution of the biological compounds is acquired by fluorescence detection. In the electrophoresis, electrodes are set in a solution such as a buffer solution, and an electric field gradient is produced in the solution by the application of direct current. At this time, when protein, DNA (Deoxyribonucleic acid), and RNA (ribo nucleic acid) having charge exist in the solution, molecules having a positive charge are attracted to a cathode and molecules having a negative charge are attracted to an anode. Thus, biomolecules can be separated.
Two-dimensional electrophoresis serving as one evaluation method using the above-described electrophoresis is an evaluation method in which biomolecules are two-dimensionally distributed in a gel by combining two kinds of electrophoresis methods, and is considered as the most effective method for proteomic analysis.
As the electrophoresis methods to be combined, for example, two kinds of methods are mainly used, that is, “electrofocusing utilizing differences in isoelectric points among individual proteins” is used as the first dimension, and “SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for separating proteins by the molecular weight” is used as the second dimension. Fluorochrome is applied to the proteins serving as the biomolecules thus separated before or after electrophoresis.
Further, an image reading device is widely spread in the fields of biochemistry and molecular biology. In the image reading device, a gel support in which the biomolecules (proteins) produced as described above are two-dimensionally distributed is irradiated with excitation light, the intensity of generated fluorescence is acquired, and an image of a fluorescence distribution (protein distribution) is displayed on the basis of the intensity.
As a method for holding the two-dimensional distribution of the biomolecules, a method is also performed, in which the biomolecules are not only held in the gel, but also transferred from the gel to a membrane by utilizing electrophoresis or a capillary action after proteins are separated in the gel. In this case, similarly to image reading using the gel support, the fluorescence distribution on a transfer support formed by the membrane can be imaged by an image reading device.
As the above-described image reading device that reads out an image of a biomolecular distribution from a gel support or a transfer support in which biomolecules are two-dimensionally distributed, Japanese Unexamined Patent Application Publication No. 10-3134 (PTL 1) discloses an image reading device.
In the above-described conventional image reading device, a mirror having a hole in its center portion is mounted on an optical head to be moved in a main scanning direction, and laser light (excitation light) with a wavelength in accordance with the wavelength of a fluorescent substance is applied through the hole of the mirror from a light source to a transfer support on which electrophoresis of denatured DNA labeled by the fluorescent substance is recorded. Then, fluorescence emitted by excitation of fluorochrome in the transfer support is reflected around the hole of the mirror, is photoelectrically converted by a multiplier, and is then detected. In this way, one line of image data is stored in a line buffer. Subsequently, by repeating the above operations while moving the optical head in a sub-scanning direction orthogonal to the main scanning direction, a two-dimensional visible image (fluorescence image) is obtained by an image processing device.
As described above, in the conventional image reading device, excitation light is applied onto the transfer support without using a dichroic mirror. Hence, compared to a method in which excitation light is applied through the dichroic mirror, greater excitation energy can be applied to the transfer support. This can increase the S/N ratio of photoelectrically detected signals (image information).
However, to detect weak fluorescence, a further increase in the S/N ratio is required. Accordingly, Japanese Unexamined Patent Application Publication No. 2000-162126 (PTL 2) discloses an image information reading device as an optical head type image reading device that provides a higher S/N ratio of detected signals than the conventional image reading device.
In this image information reading device, a mirror having a hole in its center portion is mounted on an optical head to be moved in a main scanning direction, and laser light with a wavelength for exciting fluorochrome is applied upward through the hole of the mirror from a laser light source onto a transfer support in which biogenic substances labeled by the fluorochrome are distributed. Then, fluorescence emitted downward by excitation of the fluorochrome in the transfer support reaches the mirror. In contrast, fluorescence emitted to the upper side of the transfer support is reflected by an inner surface of a concave mirror, travels downward, passes through the transfer support, and reaches the mirror in the optical head. In this way, both the fluorescences reaching the mirror are reflected around the hole of the mirror, are photoelectrically converted by a multiplier, and are then detected. Thus, one line of image data is stored in a line buffer. By subsequently repeating the above operations while moving the optical head in a sub-scanning direction orthogonal to the main scanning direction, a two-dimensional visible image (fluorescence image) is obtained by an image processing device.
By thus increasing the amount of fluorescence to be detected by the multiplier, the S/N ratio of photoelectrically detected signals (image information) can be increased.
However, the above-described conventional image information reading device has the following problems.
That is, in synchronization with movements of the optical head in the main scanning direction and the sub-scanning direction, the concave mirror disposed above the transfer support also needs to be moved in the same directions. This complicates a moving mechanism for the concave mirror.
Further, the fluorescence is emitted from the transfer support at a wide angle. To efficiently detect the fluorescence, the fluorescence emitted at the wide angle needs to be collected at the multiplier. As a method for collecting fluorescence emitted at a wide angle with as high efficiency as possible, there is a method using an objective lens having high NA (numerical aperture). However, this increases the size of a lens element.
In this case, with the increase in size of the objective lens for collecting fluorescence, the sizes of optical elements set in the way to guide the fluorescence to the multiplier, such as a reflective mirror, a laser-light cut filter, and a light collecting lens, are also increased. For this reason, in the image reading device in which the optical system including the optical head is scanned, the total size increases with the increase in size of the optical elements. Particularly when the detection system including the multiplier is scanned while being entirely mounted on the optical head, the weight of a scanning unit increases. Hence, it is feared that high-speed scanning cannot be achieved.
Further, between a first case where a gel support in which the biomolecules (proteins) are two-dimensionally distributed or a transfer support in which a pattern of the gel support is transferred on a membrane is directly placed on a glass plate to read an image of distribution of the biomolecules and a second case where a gel support in which the biomolecules (proteins) are two-dimensionally distributed is placed on the glass plate while being sealed in a two-dimensional electrophoresis substrate to read an image of distribution of the biomolecules, the distance from the objective lens to the support having the two-dimensional distribution is different by an amount corresponding to the thickness of a portion of the two-dimensional electrophoresis substrate on a lower side of the sealed support. The distance is longer in the second case than in the first case.
Therefore, when the position in the optical axis direction of the optical system including the objective lens is set to be the most suitable for the first case, fluorescence from the gel support is not collected by the multiplier in the second case. Particularly when an objective lens having high numerical aperture NA is used, the above problem is pronounced in an outer peripheral portion of the objective lens.